The N-end rule pathway is one ubiquitin (Ub) proteolytic pathway that relates the in vivo half-life of a protein to the identity of its N terminal residue. Conjugation of arginine (Arg) from Arg-tRNA(Arg) to N-terminal aspartate (Asp), glutamate (Glu), or cysteine (Cys) is part of this proteolytic pathway in that it leads to ubiquitylation of the resulting, Arg-conjugated proteins. We previously observed that ATE1-/- embryos die due to various cardiovascular defects. However, the exact nature of the cardiovascular defects and the underlying molecular mechanisms are unknown. The overall goal of this project is to determine the physiological role of ATE1-dependent arginylation in the myocardial growth and to gain insights into the cardiovascular pathways/molecules in which the ATE1-dependent proteolytic pathway participates. Functional proteomic approach to identify the substrates of the N-end rule pathway and subsequent preliminary characterization of the selected candidate substrates led to the hypothesis that RGS4, RGS5, and RGS16 are ATE1 substrates that may underlie the ATE1-dependent cardiovascular system. RGS proteins are GTPase-activating proteins (GAP) for G(alpha) subunits and negatively regulate the G protein- coupled receptor (GPCR) signaling. RGS4, RGS5, and/or RGS16 have been implicated as important negative regulators of the Gq/Gi-activated signaling for myocardial growth and vascular maturation/integrity. We then asked the mechanism by which these RGS proteins are ubiquitylated for proteolysis by ubiquitin ligases (E3s). Using an affinity-based proteomic approach, we identified a novel protein family, named UBR1-UBR7, that potentially acts for ubiquitylation of RGS4, RGS5, and RGS16. Our overall hypothesis is that the cellular concentrations of RGS4, RGS5, and RGS16, and thus their functions as regulators of the cardiovascular G protein pathways, are regulated through the MetAPs-O2-ATE1-UBR proteolytic cascade. To address this hypothesis and related issues, we propose the following Aims. Aim 1: To test whether ATE1 has an in vivo role in intrinsic myocardial growth. Aim 2: To determine the role of ATE 1-dependent arginylation for turnover of RGS4, RGS5, and RGS16. Aim 3: To elucidate the molecular mechanism by which RGS4, RGS5, and RGS16 are ubiquitylated for proteolysis. Aim 4: To examine the physiological consequences of ATE1 knockout on the G protein pathways.